Production of infectious bursal disease virus in mammalian cell line

ABSTRACT

A method of producing infectious bursal disease virus comprising culturing infectious bursal disease virus in a mammalian cell line for two to ten days.

This is a continuation of application Ser. No. 07/350,656, filed May 11,1989, now issued as U.S. Pat. No. 5,192,539.

The invention relates to the use of permanent mammalian cell lines forthe multiplication of the infectious bursal disease virus (IBDV), whichis infectious to birds, and of IBDV antigen.

Avian viruses are usually produced in embryonated eggs, on bird cellsubstrates derived from embryonated eggs, such as primary or secondarychicken embryo fibroblasts (CEF), primary chicken liver cells or chickenkidney cells, or in organs of live animals, such as in the bursa ofFabricius. Viruses from sources of this type are used throughout theworld for the preparation of inactivated and live vaccines.

The principal disadvantage in using animals and embryonated eggs for thepreparation of vaccines is the uncertainty with regard to the quality ofthese. Even specific pathogen-free chickens can unexpectedly becomeinfected, making them unsuitable for vaccine production. Occasionally aninfection of this type remains undetected for some time.

The use of a permanent cell line could provide an ideal solution to thisproblem. However, chicken cell lines, which are suitable for vaccineproduction, have not been available up to now. The majority of bird celllines consist of lymphoblastoid cells, which are obtained from animalswith lymphoid leucosis or Marek's disease.

Attempts to develop permanent cell lines from normal chicken embryofibroblasts have not proved successful. Cell lines have occasionallybeen developed from normal embryos, but in all cases these wereafterwards found to contain retrovirus genomes, and some even to shedvirus particles.

It has now been found that IBDV strains which can grow on cell culturesof chicken embryo fibroblast (such as the D78 and SP strains) can alsobe cultured efficiently in mammalian cell lines. It has been found thatit is not necessary to adapt the viruses to the mammalian substrate.Moreover, it has been found that the yields in mammalian cells arefrequently much higher than in the CEF system. Virus yields are usuallyexpressed in "infectious virus particles per unit volume" (EID₅₀ /ml;TCID₅₀ /ml). Another way to quantify virus yields is to determine theantigen mass. Using immunochemical techniques such as ELISA the antigencontent of a virus preparation can be compared with that of a standardpreparation, to which a fixed value of antigen mass units has beenassigned. With both types of method of determination, mammalian cellline systems give much higher yields than the CEF system.

Furthermore, it is surprising that these favourable yields are achievedat cell concentrations which are lower than those conventionallyemployed for antigen production on CEF. The optimum cell concentrationin the mammalian cell system is 3-6 times lower than that which iscustomarily used for production on CEF. These results show thatmammalian cells in general are better substrates for IBDV than CEF. Atthe same time it was found that the virus antigen prepared in this wayis at least as effective in a vaccine as antigen produced on CEF.

Suitable mammalian cell lines for IBDV production according to theinvention are, for example, Vero cells, chimpanzee liver cells, buffalovervet cells and mouse 3T3 cells.

Stationary culture systems in cell culture flasks and roller flasks canbe used for the culture of mammalian cells. Other, usually larger scale,cell culture systems are stirred vessels (fermenters) for the culture ofanchorage-independent cells, microcarrier systems for the culture ofanchorage-dependent cells and hollow fibre systems for the culture ofboth types of cell. In addition, there is a multiplicity of otherstationary systems for the culture of anchorage-dependent cells. Acommon feature of the latter systems is that they have a very largesurface for cell attachment.

The culture of mammalian cells requires the use of complex culturefluids. These commonly consist of a base fluid (medium), which ischemically well defined, and one or more additives, which are chemicallyless well defined. The additives are usually protein-rich solutions,such as serum and protein hydrolysis products. Serum is virtuallyindispensable for cell growth and cell division. Foetal calf serum(FoCS) or fasting calf serum (FaCS) is added to most culture systems ina concentration of 1-10% (V/V). Only in special cases it is possible,after a period of adaptation, to culture mammalian cells in serum-freeor even protein-free culture medium.

The IBD viruses according to the invention can be incorporated invaccines as live viruses, if desired after prior attenuation, or asinactivated viruses.

The vaccines containing live virus can be prepared and marketed in theform of a suspension, or lyophilized.

Lyophilized vaccines can preferably contain one or more stabilizers.Suitable stabilizers are, for example, SPGA (Bovarnik (1950): J.Bacteriology 59; 509), carbohydrates (such as sorbitol, mannitol,starch, sucrose, dextran or glucose), proteins (such as albumin orcasein), or degradation products thereof, protein-containing materials(such as bovine serum or skimmed milk) and buffers (such as alkali metalphosphates). If desired, one or more compounds with an adjuvant actioncan also be added. Suitable compounds for this purpose are, for example,aluminium hydroxide, phosphate or oxide, mineral oil (such as Bayol,Marcol 52 and saponins.

The aim of inactivation of IBD viruses is to eliminate both reproductionand virulence of the viruses. In general, this can be achieved bychemical or physical means. Chemical inactivation can be effected bytreating the viruses with, for example, enzymes, formaldehyde,β-propiolactone, ethylene-imine or a derivative thereof, an organicsolvent (such as a halogenated hydrocarbon) and/or a detergent (such asTween, Triton, sodium desoxy-cholate, sulphobetain or cetyltrimethylammonium salts). If necessary, inactivating substance isneutralized afterwards; material inactivated with formaldehyde can, forexample, be neutralized with thiosulphate. Physical inactivation canpreferably be carried out by subjecting the viruses to energy-richradiation, such as UV light, X-radiation or γ-radiation. If desired, thepH can be brought back to a value of about 7 after treatment.

Usually, an adjuvant (for example such as mentioned above), and, ifdesired, one or more emulsifiers, such as Tween and Span, is also addedto the inactivated virus material.

The vaccines according to the invention are suitable for protectingpoultry (such as chickens and turkeys) against IBD (Gumboro's disease).

The vaccines according to the invention can, for example, beadministered by means of intramuscular, subcutaneous or in ovoinjection, eyedrops, nosedrops, or drinking water or in the form ofsprays.

The invention also includes combination vaccines with the IBDV materialaccording to the invention. For inactivated vaccines, this IBDV materialcan be combined with antigen material of Newcastle Disease Virus,Infectious Bronchitis Virus, Egg Drop Syndrome Virus, Reovirus, bacteria(such as Escherichia coli) and/or parasites (such as Eimeria species).The combinations with New castle Disease Virus and/or Marek Virus arevery suitable for live combination vaccines.

EXAMPLES

Preparation of IBDV on Mammalian Cell Lines

Cell Culture

Cell stocks were stored in glass ampules in liquid nitrogen. To start acell culture the contents of an ampoule were rapidly thawed and slowlydiluted with cell culture medium. The cell suspension was centrifuged atlow speed to remove the DMSO in the refrigerant. The sedimented cellswere resuspended in complete cell culture medium and the cells wereseeded in suitable culture vats. Usually, the cells were cultured in amixture of M 199/F10 medium in a ratio of 1:1, or in MEM, supplementedwith tryptose phosphate broth. The medium contained 2-10% FoCS and, ifdesired, antibiotics and a fungicide. The cells were cultured at 37° C.in stationary culture (tissue culture flasks) or in roller flasks (490cm²). After the cells had reached a density such that they formed adense monolayer, the cells were treated with trypsin for the preparationof subcultures.

Virus Production, Harvest and Inactivation

Freeze-dried seed virus was redissolved or deepfrozen seed virus wasthawed and diluted with cell culture medium to a concentration at whichit is possible to divide the seed virus in the correct portions. Thecells were infected in a M.O.I. (multiplicity of infection) ratio of 10⁰-10⁻⁴ TCID₅₀ /cell.

If desired, seed virus was added directly after seeding the cells.

The infected cells were incubated for up to 10 days. Usually, the viruswas harvested 2-10 days after virus infection. The supernatant liquor inthe culture vats was collected and inactivated with formalin. For thispurpose, formaldehyde was added to the cell suspension until aconcentration of 0.05-0.2% was reached. The mixture was incubated for1-3 days at 20°-22° C.

Vaccine Production

For use as a vaccine, inactivated IBDV antigen was introduced into awater-in-oil emulsion (Marcol 52).

Production of IBDV on Primary Chicken Embryo Fibroblasts (CEF)

Preparation of CEFs

CEFs were prepared from 10-11-day-old, specific pathogen-free, incubatedeggs in accordance with the method known to those skilled in the art.

Culture of CEFs

CEFs were cultured in the same media as were used for culture of thecontinuous cell lines. FoCS or FaCS was added to the media until theconcentration reached 5%. A concentrated cell suspension, obtained aftertrypsin treatment, was diluted with cell culture medium to aconcentration of 0.5-10×10⁶ cells/ml. The cell suspension wastransferred to tissue culture flasks or roller flasks. The cells wereincubated for approximately 24 hours at a temperature of 37°-39.5° C.,after which a dense monolayer had formed.

Production, Harvesting and Inactivation of Virus

Freeze-dried seed virus was dissolved and diluted with cell culturemedium to a volume sufficiently large to divide into suitable amountsfor seeding on cells. The virus was added in a concentration of 10⁰-10⁻⁵ TCID₅₀ /cell. If desired, seed virus was added directly afterseeding the cells.

The infected cells were incubated for 48-96 hours. The supernatantliquor was then collected from the culture vats and inactivated with0.2% formaldehyde for 24 hours at room temperature.

Determination of IBDV Antigen Mass

IBDV antigen was determined with the aid of a quantitative sandwichELISA;

1. microtitre plates were coated with IBDV-specific antibody;

2. the wells were filled with dilution series of the antigen samples andincubated;

3. the wells were then incubated with IBDV-specific antibody which isconjugated with enzyme;

4. subsequently a substrate for the enzyme was added to the wells;

5. after some time the enzyme reaction in the wells was stopped withdilute sulphuric acid and the colour intensity of the contents of thewells determined spectrophotometrically.

The absorptions measured were compared with those of a dilution seriesof a standard antigen preparation of a known concentration. The antigenmass values were expressed in ELISA units/ml (EU/ml).

Virus Titration

Determination of the infectious virus titre was carried out on primarychicken embryo fibroblasts in microtitre plates. A ten-fold dilutionseries of the samples was first made in a primary CEF suspension whichcontains 5×10⁵ cells/ml. The wells of the microtitre plates were thenfilled with, in each case, 200 μl of the dilution series of the sample;each sample was tested 6-10 times. The plates were incubated for 4-7days at 37°-39° C. in a CO₂ incubator, after which they were examinedmicroscopically for the occurrence of cytopathic effects. The titre wascalculated in TCID₅₀ /ml. Usually, the virus titres were expressed as ¹⁰log TCID₅₀ /ml.

Determination of Virus-Neutralizing Antibodies

Virus-neutralizing antibodies were determined in a microneutralizationtest in microtitre plates. For this purpose duplicate dilution series ofthe serum samples were incubated with 1,000 TCID₅₀ of the IBDV in aserum-free cell culture medium for 1-2 hours at 37° C. in a CO₂incubator. 10⁵ primary chicken embryo cells in complete cell culturemedium were then introduced into each well. The plates were incubatedfor 4-7 days at 37° C. in a CO₂ incubator, after which they wereexamined under a microscope for the occurrence of cytopathic effects.The virus neutralization (VN) titre was now defined as the reciprocal ofthe highest dilution at which the cytopathic effect is completelyabsent. Usually, the VN titres were expressed as ² log VN titre.

EXAMPLE 1

Production of D78 Antigen on a Large Scale on Primary Chicken EmbryoFibroblasts

CEFs were obtained from 11-day-old specific pathogen-free chickenembryos. These cells were seeded in 1,585 cm² glass roller flasks in aconcentration of 1-3×10⁶ cells/ml in M199/F10 cell culture mediumsupplemented with 5% FaCS. 300 ml cell suspension were used per flask.

The flasks were incubated for 18-24 hours at 38.5°-39.5° C. Seed virusand an extra quantity of cells were then added to the flasks. 100 mlsuspension containing 3-9×10⁶ cells/ml and 10⁴ -10⁶ TCID50 of D78 seedvirus were used per roller flask. The roller flasks were then incubatedfor a further 48-120 hours at 38.5°-39.5° C. after which the virussuspension was harvested and inactivated with formaldehyde. The resultsof the antigen mass determinations on eight representative productionbatches are summarized in Table 1.

                  TABLE 1                                                         ______________________________________                                        Antigen mass (EU/ml)                                                          lowest value    highest value                                                                            mean                                               ______________________________________                                        397             1996       946                                                ______________________________________                                    

EXAMPLE 2

Preparation of D78 Antigen on Primary Chicken Embryo Fibroblasts UnderOptimized Laboratory Conditions

CEFs were obtained from 11-day-old specific pathogen-free chickenembryos. The cells were seeded in 490 cm² plastic roller flasks inconcentrations of 1.5, 3.0 and 6.0×10⁶ cell/ml in M199/F10 cell culturemedium supplemented with 5% FoCS. 100 ml cell suspension were used perflask.

At the same time 10⁷.3 TCID50 seed virus was added to each flask,resulting in infection amounts of 0.12, 0.06 and 0.03 TCID₅₀ /cellrespectively. The roller flasks were incubated at 37° C. Samples weretaken after incubating for two days and harvesting was on the third day.The antigen mass was determined using ELISA in both the samples and theharvested material. The results are given in Table 2.

                  TABLE 2                                                         ______________________________________                                                 Antigen mass Antigen mass/10.sup.6 cells                                      (EU/ml)      (EU/10.sup.6 cells)                                     Initial cell                                                                             2 days   3 days    2 days                                                                              3 days                                    concentration                                                                            p.i.     p.i.      p.i.  p.i.                                      ______________________________________                                        1.5 × 10.sup.6                                                                     2779     3218      1852  2145                                      3.0 × 10.sup.6                                                                     4353     5361      1451  1787                                      6.0 × 10.sup.6                                                                     6467     6652      1078  1109                                      ______________________________________                                    

Conclusion: under optimized laboratory conditions it is possiblesubstantially to improve the production of antigen mass on CEF, but theproduction per cell is considerably reduced when the cell concentrationis increased.

EXAMPLE 3

Preparation of IBDV Antigens on Vero Cells

A. Infection on a fully grown monolayer

Vero cells were seeded in two 490 cm² roller flasks in a concentrationof 0.25×10⁶ cells/ml. 100 ml cell suspension in Eagle's MEM supplementedwith tryptose phosphate broth and 5% FoCS were added to each rollerflask. One flask was incubated for 4 days and the other for 5 days at37° C. The cell culture medium was then removed and D78 seed virus wasadded in a quantity of 10⁶ TCID₅₀ per roller flask. After incubation ofthe cell-virus mixture for 30 minutes at 37° C., 100 ml of cell culturemedium were added. After a further incubation for eight or seven daysrespectively, the virus suspension was harvested and the antigen massdetermined. This was found to be 19413 EU/ml after seven days'incubation and 19706 EU/ml after eight days.

B. Infection of cells with IBDV in suspension

Vero cells were seeded in 490 cm² plastic roller bottles in aconcentration of 1×10⁶ cells/ml in Eagle's MEM supplemented withtryptose phosphate broth and 5% FoCS. 100 ml of cell suspension wereused per flask. The IBD seed virus, strain D78 or SP, was also addedimmediately after the cells.

The cultures were incubated for 7 days. Samples were taken 45, 96 and144 hours after infection. The antigen mass contents of these sampleswere determined with the aid of ELISA (Table 3).

                  TABLE 3                                                         ______________________________________                                                                        Antigen mass/                                                  Antigen mass   10.sup.6 seeded                               Virus M.O.I.     (EU/ml) after: cells (EU/10.sup.6                            strain                                                                              TCID.sub.50 /cell                                                                        45 h   96 h  144 h cells) after 144 h                        ______________________________________                                        D78   10.sup.-1  9044   20737 22937 22937                                     D78   10.sup.-2  1197   20016 17759 17739                                     D78   10.sup.-3   161   13224 18233 18233                                     SP    10.sup.-1  3543   28439 26883 26883                                     SP    10.sup.-2   509   21799 23165 23165                                     SP    10.sup.-3    64   15121 13934 13934                                     ______________________________________                                    

Conclusion: production of IBDV on mammalian cells yields antigen massesin approximately ten times the yield obtained by production on CEF undercomparable conditions in accordance with Example 2. Approximately equalproduction levels were achieved on monolayers and in suspension culture.

EXAMPLE 4

Infectious Virus Titre of IBDV Produced on Vero Cells

Vero cells were seeded in 490 cm² roller flasks in a concentration of1×10⁶ cells/mi. 100 ml cell suspension in Eagle's MEM supplemented withtryptose phosphate broth and 5% FoCS were introduced into each rollerflask. At the same time, D78 or SP seed virus was added in an amount of0.01 TCID₅₀ /cell. The flasks were incubated at 37° C. During incubationsamples were taken and the antigen mass and infectious virus containedtherein determined. The antigen mass was determined (EU/ml) using ELISA.The infectious virus titre was determined in a microtitre plate testwith the aid of primary CEFs (¹⁰ log TCID₅₀ /ml) The results are givenin Table 4:

                  TABLE 4                                                         ______________________________________                                        Yield                                                                         45 hours p.i.  96 hours p.i.                                                                              144 hours p.i.                                                  .sup.10 log    .sup.10 log  .sup.10 log                         Virus         TCID.sub.50 /  TCID.sub.50 /                                                                              TCID.sub.50 /                       strain                                                                              EU/ml   ml       EU/ml ml     EU/ml ml                                  ______________________________________                                        SP    1197    8.9      20016 10.2   17739 9.9                                 D78    509    8.2      21799 9.7    23165 9.5                                 ______________________________________                                    

EXAMPLE 5

Live Vaccine

Two groups of four SPF chickens (age three weeks) were vaccinated on day0 with live IBD virus, strain D78, produced on Vero cells. The virus wasadministered ocularly to the birds in a volume of 0.1 ml. One groupreceived 10⁶ TCID₅₀ per bird and the other 10⁴.5 TCID₅₀ per bird. Inaddition, a group of four birds was vaccinated in the same way with aD78 vaccine produced on primary CEFs. Blood was taken sixteen days aftervaccination. This was examined for the presence of IBDV-neutralizingantibodies.

The result is given in Table 5.

                  TABLE 5                                                         ______________________________________                                                 Dose           Serum neutralization                                  Vaccine  (.sup.10 log TCID.sub.50 /bird                                                               titre (.sup.2 log VN)                                 ______________________________________                                        D 78/Vero                                                                              6.0               9.1 ± 1.5 1)                                    D 78/Vero                                                                              4.5            10.6 ± 1.4                                         D 78/CEF 5.7            10.2 ± 1.8                                         ______________________________________                                         1) .sup.2 log VN ± standard deviation                                 

The same experiment also involved a number of birds from which the bursawas removed 3, 6 and 16 days after vaccination and examinedhistologically for acute and subacute lesions. No discrepancies werefound between bursas from birds vaccinated with virus produced on CEFand those of birds vaccinated with virus produced on Vero cells.

Conclusion: live IBD vaccine produced on Vero cells is just asimmunogenic and just as harmless as live IBD vaccine produced on CEF.

EXAMPLE 6

Preparation of IBDV Strain D78 in a Chimpanzee Liver Cell Line

100 ml of a suspension of chimpanzee liver cells (concentration 0.6×10⁶cells/ml) were seeded in a 490 cm² roller flask. IBDV seed virus (strainD78) was added in a concentration of 10⁻⁴ TCID₅₀ /cell. After incubatingfor seven days, the antigen mass content was 27042 EU/ml, correspondingto approximately 45×10³ EU/10⁶ seeded cells.

Example 7

Preparation of IBDV strain D78 in a Mouse Cell Line

100 ml of a suspension of NIH3T3 mouse cells (concentration 0.3×10⁶cell/ml) were seeded in a 490 cm² roller flask, after which IBD seedvirus (strain D78) was added in a concentration of 10⁻⁴ TCID₅₀ /cell.After incubating for seven days, the antigen mass content of the viruswas 3514 EU/ml, which corresponds to approximately 12×10³ EU/10⁶ seededcells.

EXAMPLE 8

Comparison of the Immunogenicity of IBDV Antigens Cultured in CEF orVero Cells

D78 and SP antigens prepared in primary CEFs or in Vero cells wereinactivated with formalin and emulsified in mineral oil. Four-week-oldspecific pathogen free chickens (white Leghorns) were each vaccinatedintramuscularly with 0.5 ml of one of the abovementioned emulsions. Sixweeks after vaccination blood was taken from these chickens, the serawere inactivated for 30 minutes at 56° C. and the virus-neutralizingantibodies contained therein were determined on primary CEFs. Theresults are summarized in Table 6.

                  TABLE 6                                                         ______________________________________                                        Virus   Production                                                                              Antigen    Neutralizing antibody                            antigen cell      mass       titre (1) (.sup.2 log VN)                        ______________________________________                                        D78     CEF       2924       14.4 ± 1.3                                    D78     CEF       1072       13.7 ± 1.7                                    D78     Vero      2750       14.6 ± 1.5                                    D78     Vero      690        13.1 ± 1.9                                    SP      Vero      4000       14.6 ± 1.1                                    SP      Vero      1000       13.5 ± 1.3                                    ______________________________________                                         (1) mean for 10 birds standard deviation                                 

EXAMPLE 9

Comparison of the Effectiveness of Vaccination with IBDV AntigensCultured in CEF or Vero Cells

Eight-week-old chickens were vaccinated in groups of 10 with inactivatedD78-oil emulsion vaccine. 0.5 ml vaccine was administeredintramuscularly to each bird. One group of birds was vaccinated withantigen produced on Vero cells and two groups with antigen produced onCEF. Both antigens were produced in roller flasks in accordance with theprocedure described in Examples 2 and 3. Six weeks after vaccination,serum was collected from the birds and examined for virus-neutralizingantibodies. The results are summarized in Table 7.

                  TABLE 7                                                         ______________________________________                                        Vaccine     .sup.2 log VN ± stand. dev.                                    ______________________________________                                        D78/CEF1    11.2 ± 1.4                                                     D78/CEF2    13.1 ± 0.9                                                     D78/Vero    14.5 ± 1.7                                                     ______________________________________                                    

Statistic analysis showed that the titre induced by D78/Vero wassignificantly higher than that induced by D78/CEF (Student's T test;p<0.05 of CEF2).

We claim:
 1. A method for the production of inactivated infectiousbursal disease virus (IBDV), comprising:(a) culturing IBDV in amammalian cell line for two to ten days; (b) harvesting the IBDV fromthe cultured cell line; and (c) inactivating the harvested IBDV by achemical inactivating substance or physical inactivation.
 2. The methodof claim 1, further comprising neutralizing the chemical inactivatingsubstance after step(c).
 3. The method of claim 1, further comprisingadjusting the pH in the inactivated IBDV to about
 7. 4. The method ofclaim 1, further comprising adding to the inactivated IBDV at least onemember selected from the group consisting of a pharmaceuticallyacceptable carrier, a diluent, an adjuvant and an emulsifier.
 5. Themethod of claim 1, wherein the cell line is an ape cell line.
 6. Themethod of claim 5, wherein the ape cell line is a Vero cell line.
 7. Themethod according to any one of claims 1-6 wherein the IBDV is strainD78.